Neutrophil responses are a critical element in host defense during bacterial infections, for example, but neutrophil responses can also be overtly pathogenic. Thus, during many inflammatory diseases neutrophils contribute more to the pathology than do the microbes themselves (Nathan, 2002, Nature 420:846-852). Neutrophil recruitment is a complex process involving activation of local structural cells (e.g., epithelial cells) via pathogen-associated molecular pattern receptors, complement cascade products or arachidonic acid metabolites, such that the structural cells express inflammatory mediators (e.g., IL-1, CXCL8) (Smith et al., 2001, J. Immunol. 167:366-374). These can in turn activate regional endothelial cells, which actively foster intravascular neutrophil retention via the now classical paradigm of chemokine- (e.g., CXCL8) and adhesion molecule-mediated rolling-arrest-diapedesis (Baggiolini, 1998, Nature 392:565-568; Springer, 1994, Cell 76:301-314).
The ELR-CXC chemokines are a subgroup of the CXC chemokine family in which the amino sub-terminal two cysteine residues, which are separated by an alternate amino acid, are immediately preceded by a Glu-Leu-Arg motif. They include CXCL1-3 and 5-8 (growth-related oncogene α, β, and γ [GRO α, β, and γ], epithelial cell neutrophil-activating peptide-78 [ENA-78], granulocyte chemotactic protein-2 [GCP-2], neutrophil activating peptide-2 [NAP-2], and interleukin-8 [IL-8], respectively)(3), which chemoattract and activate neutrophils via two closely related G protein-coupled receptors (GPCR), the CXCR1 and CXCR2. CXCL8 binds both receptors with high affinity, while CXCL6 binds both receptors with lower affinity (Wolf et al., 1998, Eur. J. Immunot 28:164-170; Wuyts et al., 1998, Eur. J. Biochem. 255:67-73). The other ELR-CXC chemokines bind to the CXCR2, also with relatively lower affinities (Wuyts et al., 1998; Ahuja and Murphy, 1996, J. Biol. Chem. 271:20545-20550). Both the CXCR1 and CXCR2 can trigger chemotactic responses and intracellular Ca2+ flux in neutrophils and contribute to elastase release (Wuyts et al., 1998; Chuntharapai and Kim, 1995, J. Immunol. 155:2587-2594), but activation of the respiratory burst and phospholipase D release responses are reportedly CXCR1-dependent (Jones et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:6682-6686), whereas CXCR2 signaling is critical to MMP-9 release (Chakrabarti and Patel, 2005, J. Leukoc. Biol. 78:279-288). Moreover, these two receptors may be differentially involved in neutrophilic pathology in vivo. For example, the CXCR1 is reportedly of more importance in inflammatory bowel diseases (Gijsbers et al., 2004, Eur. J. Immunol. 34:1992-2000), sepsis and acute respiratory distress syndrome (Cummings et al., 1999, J. Immunol. 162:2341-2346; Goodman et al., 1999, Chest 116:111s-112s), while both it and the CXCR2 play roles in synovial infiltration by neutrophils in arthritic joints (Podolin et al., 2002, J. Immunol. 169:6435-6444). However, numerous alternate GPCR are involved in neutrophilic inflammation, including those for LTB4, C5a, and fMLP, such that antagonism of either LTB4 or C5a has been shown to be of significant benefit in various inflammatory settings (Park et al., 2000, Anesth. Analg. 89:42-48; Crooks et al., 2000, Eur. Respir. J. 15:274-280; Wollert et al., 1993, Surgery 114:191-198). The precise interrelationships of these mediators in inflammatory responses have not been formally determined, but it has been reported that signaling through the C5a or fMLP receptors can effectively desensitize the ELR-CXC chemokine receptors (Blackwood et al., 1996, J. Leukoc. Biol. 60:88-93). On the other hand, CXCL8 reportedly poorly desensitizes some events (e.g., intracellular Ca++ flux) associated with C5a and fMLP receptor signaling (Richardson et al., 1995, J. Biol. Chem. 270:27829-27833; Tomhave et al., 1994, J. Immunol. 153:3267-3275), although it can desensitize chemotactic responses driven by these ligands.
Pulmonary aspiration, brought on by aspiration of gastric contents into the lungs, occurs primarily in unconscious or semiconscious patients. Its incidence is approximately 1 in 3000 during general anaesthesia surgeries (Olsson et al., 1986, Acta Anaesthesiol Scand. 30(1):84-92; Warner et al., 1993, Anesthesiology 78(1):56-62).
The local inflammatory sequel varies from sub-clinical pneumonitis to severe acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (Pepe et al., 1982, Am J Surg 144(1):124-30; Fowler et al., 1983, Ann Intern Med 98(5 Pt 1):593-7) depending on the volume and the pH of the gastric contents (Marik, 2001, N Engl J Med 344(9):665-71). However, ALI/ARDS associated with aspiration pneumonia carries a mortality rate of 10-30% (Olsson et al., 1986; Warner et al., 1993).
Neutrophils are the primary drivers of the inflammatory cascade in aspiration pneumonia (Beck-Schimmer et al., 2005, Anesthesiology 103(3):556-66; Raghavendran et al., 2005, Am J Physiol Lung Cell Mol Physiol 289(1):L134-43; Folkesson et al., 1995, J Clin Invest 96(1):107-16; Davidson et al., 2005, Am J Physiol Lung Cell Mol Physiol 288(4):L699-708).
When activated, neutrophils release an array of microbicidal factors, including reactive oxygen intermediates (ROI), defensins, and proteolytic enzymes, but they also foster the inflammatory responses through elaboration of proinflammatory cytokines (e.g. TNF, IL-1) and chemokines (e.g. CXCL8/IL-8, CXCL1/GROα). The CXC chemokines that contain a glutamic acid-lysine-arginine (ELR) motif just proximal to their amino sub-terminal CXC motif (the ELR-CXC chemokines; e.g., CXCL8/IL-8, macrophage-inflammatory protein-2 [MIP-2]) are central to the recruitment and activation of neutrophils in inflammatory settings such as aspiration pneumonia (Beck-Schimmer et al., 2005; Folkesson et al., 1995; Rotta et al., 2004, Crit Care Med 32(3):747-54). This suggests that ELR-CXC chemokine antagonism could be an ideal therapeutic approach to ameliorate aspiration pneumonia pathology.
Previously, we generated a broad-spectrum ELR-CXC chemokine antagonist bovine CXCL8(3-74)K11R/G31P (bG31P) (Li and Gordon, 2001, Biochem Biophys Res Commun 286(3):595-600; Li et al., 2002, Biochem Biophys Res Commun 293(3):939-44), which blocks the ability of ELR-CXC chemokines to activate and chemoattract neutrophils in vitro (Li et al., 2002). A single treatment with bG31P≈97% blocks neutrophil infiltration into intradermal endotoxin challenge sites for 2-3 days (Li et al., 2002, Vet Immunol Immunopathol 90(1-2):65-77), and dramatically reduces pulmonary pathology and pyrexia in endotoxemic animals (Gordon et al., 2005, J Leukoc Biol 78(6):1265-72). In the process of generating a human homologue of bG31P we developed multiple human-bovine chaemeric forms of G31P that were also effective in antagonizing ELR-CXC chemokine-mediated pathology (Xixing Zhao et al., 2007, International Immunopharmacology 7:1723-31), and more recently we engineered a fully human form of G31P, human CXCL8(3-72) K11R/G31P (G31P). This latter drug not only blocks neutrophil activation induced by ELR-CXC chemokines, but also human airway epithelial cell responses driven by bacterial lipopolysaccharide (LPS). Importantly, it also partially antagonizes heterologous GPCR that are involved in neutrophilic inflammation, including those for C5a, LTB4, and fMLP. In the present study, we characterized the effects of this antagonist on blocking neutrophil-mediated acute lung injury caused by aspiration of bacterial-laden, acidified gastric contents. A primary goal was to determine whether antagonizing the neutrophil response would negatively impact bacterial clearance in the lungs of the affected animals.
It has been widely accepted that intestinal ischemia/reperfusion (I/R) injury is associated with severe multiple organ failure (MOF), including the lung (He et al., 2008, PLoS ONE 3: e1527; Schmeling et al., 1989, Surgery 106: 195-201; Caty et al., 1990, Ann Surg 212: 694-700). A number of proinflammatory cytokines and chemokines are critically involved in this pathology (Caty et al., 1990) although the pathogenesis of intestinal I/R-induced gut pathology and MOF is complex and not completely understood. Nonetheless, reactive oxygen intermediate (ROI) generation and neutrophil sequestration are believed to be two fundamental causative factors (Schmeling et al., 1989; Cerqueira et al., 2005, Acta Cir Bras 20: 336-343; Souza et al., 2004 Br J Pharmacol 143: 132-142). The pivotal role of neutrophils in these processes (Schmeling et al., 1989; Cerqueira et al., 2005; Bless et al., 1999, Am J Physiol 276: L57-63) suggests that blockade of their recruitment could be an important therapeutic approach to intestinal I/R injury. The list of neutrophil agonists potentially involved in I/R injury includes TNFα and IL-1β (Caty et al., 1990) and the ELR-CXC chemokines (e.g., CXCL1, CXCL8, MIP-2) (Schmeling, et al., 1989; Bless et al., 1999; Sekido et al., 1993, Nature 365: 654-657), but also C5a (Bless et al., 1999; Wada et al., 2001, Gastroenterology 120: 126-133), LTB4 (Souza et al., 2000, Eur J Pharmacol 403: 121-128), ROI (Koike et al., 1993, J Surg Res 54: 469-473), and adhesion molecules (Bless et al., 1999). Multiple reports have implicated individual ELR-CXC chemokines as primary effectors and shown that their neutralization ameliorates I/R-induced local and remote organ pathology (Souza et al., 2004; Bless et al., 1999; Miura et al., 2001, Am J Pathol 159: 2137-2145; Kaneko et al., 2007, Eur Surg Res 39: 153-159), making these mediators attractive therapeutic targets.
We have shown that G31P antagonizes CXCR1-dependent responses as well as those related to CXCR2-exclusive ligands but, importantly, it also antagonizes heterologous GPCRs (i.e., C5a, LTB4, and fMLP) that are involved in neutrophilic inflammation. We hypothesize that G31P's ability to antagonize both ELR-CXC chemokine receptors as well as these alternate GPCR could potentially provide it with a strong therapeutic advantage during neutrophilic inflammatory events. In this study, we employed a rat model of superior mesenteric artery (SMA) ischemia and reperfusion (I/R) injury and characterized the effects of G31P on local and remote organ pathology in this model system.
Some of the protective effects of bG31P in airway endotoxemia, for example, seemed to be somewhat tangentially related to its putative neutrophil-centered effects. For example, bG31P treatment reduces endogenous pyrogen expression and pyrexia prior to the time when neutrophils were appreciably present in the airways, suggesting that it may have effects on structural (e.g., epithelial) cells. Moreover, data from a study of its effects in aspiration pneumonia, wherein high level bacterial colonization of the lungs occurs, confirmed its effectiveness in this environment as well. In such cases the bacteria would release formyl peptides (e.g., fMLP) among other products and activate the complement cascade, leading to the generation of C5a. Thus, we wished to explore further the mechanisms by which G31P might interact with airway epithelial cells, but also its effect on neutrophil responses to ligands for heterologous GPCR.
Melanomas are frequently aggressive tumours, due primarily to their ability to metastasize via the vasculature or lymphatics to regional lymph nodes or tissues (e.g, lungs, liver). Despite our efforts to understand the development of metastases, to date our most effective treatment for melanomas remains surgical tumour removal, although radiotherapy and chemotherapy also play a significant role in treatment. The oncogenic process consists of melancocyte (de)differentiation, proliferation, vascularization, and eventual metastasis.
Herein, we tested whether ELR-CXC chemokine antagonism with G31P would by itself be beneficial in a model of melanoma. We assessed the impact of G31P on chemokine-driven tumour cell proliferation in vitro. The tumour cells were injected i.v. into the mice, which were treated at the time of challenge or at varying times thereafter with G31P. We assessed the development of tumour masses and markers in the lungs of the challenged animals